The present invention is concerned with cancer treatment and diagnosis, especially with a melanoma associated antigen, epitopes thereof, vaccines against melanoma, tumor infiltrating T lymphocytes recognizing the antigen and diagnostics for the detection of melanoma and for the monitoring of vaccination.
Tumor cells may emancipate themselves from restrictive growth control by oncogene activation, and/or by the inactivation of tumor suppression genes. The course of tumor progression proceeds by a series of gradual, stepwise changes in different xe2x80x98unit characteristicsxe2x80x99, i.e. phenotypic traits, many of which are known to be determined or at least influenced by the altered expression of defined oncogenes and/or tumor suppressive genes. Emancipation of the cell from immunological host restriction may follow multistep pathways similar to the emancipation from growth control.
A problem often encountered in cancer immunotherapy is the lack of immunogenicity of the tumor. This escape of the immune control system can be understood on basis of phenotype differences encountered in neoplastic cells (differences found in Burkitt""s lymphoma cells according to Klein, G. and Boon, T., Curr. Opinion in Immunol. 5, 687-692, 1993):
decreased ability to process and present antigens;
decreased ability to stimulate autologous T cells;
complete downregulation of immunogenic proteins associated with transformed cells;
no or low expression of leukocyte adhesion molecules or other accessory molecules; and
selective downregulation of certain MHC class I and class II alleles.
MHC Class I/II antigens are often downregulated in solid tumors. This may affect all class I/II antigens, or only part of them. Viral and cellular peptides that can sensitize appropriate target cells for cytotoxic T lymphocyte mediated lysis may fail to do so when produced in cells with a low level of expression of MHC class I antigen. Cytotoxic sensitivity may be induced, at least in some cases by raising the level of MHC class I/II antigen expression by interferon xcex3 and tumor necrosis factor xcex1.
However, during the stepwise changes from normal to tumor tissue tumor-associated antigens appear. These antigens can be exposed through various mechanisms:
they can be molecules that are masked in some way during normal cell development, but where the neoplastic change induces removal of the masking protection for the immunosystem;
deletion of some molecules from the plasma membrane may alter the profile of adjacent molecules in a given membrane patch, and thus, in effect generate a new profile that might become immunogenic to the host;
a membrane alteration accompanying neoplastic transformation may expose new, previously hidden regions of a molecule, or may result in addition of new structural features to an existing molecule.
shedding and disintegration of tumor cells may expose the immune system to nuclear, nucleolar, or cytoplasmic components that are normally hidden in the cell.
The characteristics of tumor-associated antigens are very much dependent on the origin of the tumor carrying them. The existence of antigens associated with animal tumors was documented in the last century, and the antigenic character of human cancers has been well established, primarily through recent studies with monoclonal antibodies.
Attempts to isolate and chemically characterize these antigens have encountered serious difficulties, many having to do with a lack of reagents suitable for precipitation of the antigen-bearing molecules from a solution.
Like many other stimuli, the tumor-associated antigens activate not one but a whole set of defense mechanismsxe2x80x94both specific and unspecific, humoral and cellular. The dominant role in in vivo resistance to tumor growth is played by T lymphocytes. These cells recognize tumor-associated antigens presented to them by antigen presenting cells (APC""s), and will be activated by this recognition, and upon activation and differentiation, attack and kill the tumor cells. A special class of these sort of lymphocytes is formed by the tumor infiltrating lymphocytes (TIL""s) which can be found in solid tumors.
It has already been suggested (EP 147,689) to activate T lymphocytes with an antigenic substance linked to an insoluble carrier in vitro and then to administer these activated lymphocytes to a tumor patient.
Conventional chemotherapy is relatively ineffective in the treatment of patients with metastasic melanoma, and approximately 6000 patients die of this disease in the United States each year.
Rosenberg et al. (New Eng. J. Med. 319(25), 1676-1681, 1988) have shown the beneficial effect of immunotherapy with autologous TIL""s and interleukin-2 (IL-2) in melanoma patients.
This therapy constitutes of resection of the tumor deposit, isolation of the TIL""s, in vitro expansion of the TIL""s and infusion into the patient under concurrent treatment of high and toxicity inducing doses of IL-2.
The TIL""s used by Rosenberg are directed to and able to recognize melanoma-associated antigens.
It has been our goal to isolate such a melanoma-associated antigen in order to be able to use the antigen and/or its epitopes for the development of an immunotherapy for melanoma patients.
Melanoma antigens have already been described by Old, L. (1981) who identified 6 antigenic glycoproteins and 3 glycolipids occurring in 120 melanoma cell lines.
Also vaccines with melanoma antigens have been described: in U.S. Pat. Nos. 5,030,621 and 5,194,384 a polyvalent vaccine has been made by culturing melanoma cells and subsequent isolation of excreted melanoma-specific antigens from the culture medium.
Some specific antigens have already been proposed for therapy and diagnosis of melanoma type of cancer: the peptide p97 has been disclosed in U.S. Pat. Nos. 5,262,177 and 5,141,742, while a 35 kD protein has been mentioned in EP 529,007.
We now have found a melanoma-associated polypeptide, characterized in that it comprises the aminoacid sequence of SEQ ID NO: 2.
This melanocyte lineage-specific antigenic polypeptide (also mentioned gp100) is recognized by the monoclonal antibody NKI-beteb, which antibody has proven suitable for diagnostic purposes. The antigens recognized by this antibody are intracellular proteins of approximately 10 kd (gp 10) and 100 kd (gp100). The latter is also detectable in a culture medium of melanoma cells (Vennegoor, C. et al, Am. J. Pathol. 130, 179-192, 1988). It has also been found that the gp100 antigen reacts with other melanoma-specific antibodies such as HMB-50 (described by Vogel, A. M. and Esclamado, R. M., Cancer Res. 48, 1286-1294, 1988) or HMB-45 (described by Gown, A. M. et al., Am. J. Pathol. 123, 195-203, 1986). Since the proteins reacting with these monoclonal antibodies have been shown te be glycosylated in melanoma cells, differences have been found in mobility when analyzed by SDS-PAGE.
Although this gp100 antigen is predominantly expressed intracellularly, it has now been established that it is a suitable immunogenic antigen, because it has been demonstrated that these intracellular proteins can be processed and presented as peptides in the context of MHC molecules to cells of the immune system. In fact, tumor infiltrating lymphocytes derived from tumors of melanoma patients have been found which react with the antigen.
Therefore, the gp100 polypeptide is a potential target for cellular responses against carcinoma and thus a suitable subject for therapy and diagnosis in melanoma patients.
Gp100 is a type I transmembrane protein, which has a threonine-rich domain containing repetitive amino acid sequences present in the middle of the protein (amino acids 309-427). This threonine-rich domain, which may be subjected to extensive O-linked glycosylation, is preceded by a histidine-rich region (amino acids 182-313) and followed by a cysteine-rich domain (amino acids 475-566). Based on hydrophobicity plot analysis (Kyte, J. and Doolittle, R. F., 1982), a single transmembrane domain bordered by charged residues is present in the carboxy-terminal part (amino acids 591-611) of gp100. The predicted cytoplasmic domain is 45 amino acids long. Five putative N-linked glycosylation sites are present, consistent with gp100 being a glycoprotein.
The term xe2x80x9cpolypeptidexe2x80x9d refers to a molecular chain of amino acids, does not refer to a specific length of the product and if required can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation; thus inter alia peptides, oligopeptides and proteins are included within the definition of polypeptide.
Of course, functional derivatives as well as fragments of the polypeptide according to the invention are also included in the present invention. Functional derivatives are meant to include polypeptides which differ in one or more amino acids in the overall sequence, which have deletions, substitutions, inversions or additions. Amino acid substitutions which can be expected not to essentially alter biological and immunological activities, have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science 227, 1435-1441, 1985) and determining the functional similarity between homologous polypeptides.
Functional derivatives which still show immunological activity towards the monoclonal antibody NKI-beteb or HMB-50 or HMB-45 are included within the scope of this invention.
Furthermore as functional derivatives of these peptides are also meant peptides derived from gp100 which are able to induce target cell lysis by tumor infiltrating lymphocytes.
In addition, with functional derivatives of these peptides are also meant addition salts of the peptides, amides of the peptides and specifically the C-terminal amides, esters and specifically the C-terminal esters and N-acyl derivatives specifically N-terminal acyl derivatives and N-acetyl derivatives.
The polypeptides according to the invention can be produced either synthetically or by recombinant DNA technology. Methods for producing synthetic polypeptides are well known in the art.
The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a condensation reaction, either in homogenous phase or with the aid of a so-called solid phase. The condensation reaction can be carried out as follows:
a) condensation of a compound (amino acid, peptide) with a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) with a free amino group and protected other reactive groups, in the presence of a condensation agent;
b) condensation of a compound (amino acid, peptide) with an activated carboxyl group and free or protected other reaction groups with a compound (amino acid, peptide) with a free amino group and free or protected other reactive groups.
Activation of the carboxyl group can take place, inter alia, by converting the carboxyl group to an acid halide, azide, anhydride, imidazolide or an activated ester, such as the N-hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl ester.
The most common methods for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.).
Production of polypeptides by recombinant DNA techniques is a general method which is known, but which has a lot of possibilities all leading to somewhat different results. The polypeptide to be expressed is coded for by a DNA sequence or more accurately by a nucleic acid sequence.
It has been found that the amino acid sequence of gp100 closely resembles the amino acid sequence of the already known melanoma-associated peptide pMel17, disclosed in Kwon, B. S. (1991).
The amino acid differences between gp100 and Pmel17 consist of substitutions at amino acid position 274 (T-C/PRO-LEU) and 597 (C-G/ARG-PRO) and a stretch of 7 amino acid absent in gp100 at position 587. A single nucleotide difference at position 762 (C-T) does not result in an amino acid substitution. Gp100 is also 80% homologous to a putative protein deduced from a partial cDNA clone (RPE-1) isolated from a bovine retinal cDNA library (Kim, R. Y. and Wistow, G. J., 1992) and 42% homologous to a chicken melanosomal matrix protein, MMP115 (Mochii, M., 1991). See also FIG. 2.
Also part of the invention is the nucleic acid sequence comprising the sequence encoding the gp100 polypeptide.
Preferably the sequence encoding gp100 is the sequence of SEQ ID NO:1.
As is well known in the art, the degeneracy of the genetic code permits substitution of bases in a codon resulting in another codon still coding for the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of a polypeptide with an amino acid sequence shown in SEQ ID NO:2 use can be made of a derivate nucleic acid sequence with such an alternative codon composition thereby differring from the nucleic acid sequence shown in SEQ ID NO:1.
xe2x80x9cNucleotide sequencexe2x80x9d as used herein refers to a polymeric form of nucleotides of any length, both to ribonucleic acid (RNA) seqeuences and to desoxyribonucleic acid (DNA) seqeuences. In principle this term refers to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, as well as double and single stranded RNA, and modifications thereof.
The nucleotide sequence of gp100 contains 2115 base pairs (bp) and terminates with a poly (A) tract of 15 nucleotides which is preceded by the consensus polyadenylation sequence AATAAA SEQ. ID. NO. 31. An open reading frame (ORF) extending from nucleotide 22 through 2007 is present in gp100 DNA. This ORF starts with an ATG codon within the appropriate sequence context for translation initiation and codes for a protein of 661 amino acids. The amino-terminal 20 amino acids fit all criteria for signal sequences, including a potential cleavage site after ALA at position 20 (xe2x88x921), which would indicate that mature gp100 contains 641 amino acids (approximately 70 kD).
The most striking difference between gp100 and Pmel17 cDNAs is the inframe deletion of 21 bp in gp100 cDNA (FIG. 2). Comparison of the nucleotide sequence of genomic DNA with the sequence of gp100 cDNA revealed the presence of an intron (102 bp) just at the position of the 21 bp insertion in Pmel17 cDNA. The exon/intron boundaries nicely fit the consensus 5xe2x80x2 donor and 3xe2x80x2 acceptor splice site sequences (Padgett, 1986). In the genomic DNA, the sequence comprising the additional 21 bp in Pmel17 cDNA is located directly upstream of the 3xe2x80x2 cleavage site used to generate gp100 RNA and is preceded by an alternative 3xe2x80x2 acceptor splice site. Whereas the gp100-specific 3xe2x80x2 acceptor splice site fits the consensus sequence, the Pmel17-specific 3xe2x80x2 acceptor splice site appears to be sub-optimal in that it lacks a pyrimidine-rich region. Sub-optimal RNA processing sites are present in many alternatively processed messenger RNA precursors and have been implicated to function in regulation of alternative RNA processing (reviewed by Green, M. R., 1991). Collectively, these data prove that the transcripts corresponding to gp100 and Pmel17 cDNAs are generated by alternative splicing of a single primary transcript.
A further part of the invention are peptides, which are immunogenic fragments of the gp100 polypeptide.
Immunogenic fragments are fragments of the gp100 molecule, which still have the ability to induce an immunogenic response, i.e. that it is either possible to evoke antibodies recognizing the fragments specifically, or that it is possible to find T lymphocytes which have been activated by the fragments.
As has been said above it has been known that the immunogenic action of tumor associated antigens is often elicited through a T cell activating mechanism (Townsend, A. R. M. and Bodmer, H., Ann. Rev. Immunol. 7, 601-624, 1989). Cytotoxic T lymphocytes (CTLs) recognizing melanoma cells in a T cell receptor (TCR)-dependent and MHC-restricted manner have been isolated from tumor-bearing patients (reviewed by Knuth, A., 1992). Brichard et al. (1993) have shown that a peptide derived from tyrosinase, an other melanocyte-specific antigen, is recognized by a CTL clone.
It is known that the activation of T cells through the MHC molecule necessitates processing of the antigen of which short pieces (for example 8-12 mers) are presented to the T lymphocyte.
The immunogenic oligopeptides located in the gp100 sequence form also part of the invention.
We have found immunogenic peptide sequences of the gp100 sequence which are not only able to bind with the MHC I molecule, but which also have been demonstrated to recognize tumor infiltrating lymphocytes which have been isolated from a melanoma patient.
Several peptides have been found: the peptides having the amino acid sequences V-L-P-D-G-Q-V-I-W-V (SEQ ID NO:5), M-L-G-T-H-T-M-E-V (SEQ ID NO:24), R-L-M-K-Q-D-F-S-V (SEQ ID NO:25), (V)-(W)-(K)-T-W-G-Q-Y-W-Q-V-(L) (SEQ ID NO:10) and L-L-D-G-T-A-T-L-R-L (SEQ ID NO:4) have been found to bind to the MHC HLA-A2.1 molecule. In addition, the latter two peptides are recognized by anti-melanoma cytotoxic T lymphocytes in the context of HLA-A2.1.
Preferably these peptides are flanked by non-related sequences, i.e. sequences with which they are not connected in nature, because it has been found that such flanking enhances the immunogenic properties of these peptides, probably through a better processing and presentation by APC""s.
Another part of the invention is formed by nucleotide sequences comprising the nucleotide sequences coding for the above mentioned peptides.
Next to the use of these sequences for the production of the peptides with recombinant DNA techniques, which will be exemplified further, the sequence information disclosed in the sequence listings for gp100 or its epitopes can be used for diagnostic purposes.
From these sequences primers can be derived as basis for a diagnostic test to detect gp100 or gp100-like proteins by a nucleic acid amplification technique for instance the polymerase chain reaction (PCR) or the nucleic acid sequence based amplification (NASBA) as described in U.S. Pat. No. 4,683,202 and EP 329,822, respectively.
With PCR large amounts of DNA are generated by treating a target DNA sequence with oligonucleotide primers such that a primer extension product is synthesized which is separated from the template using heat denaturation and in turn serves as a template, resulting in amplification of the target sequence. When RNA is to be amplified with PCR the RNA strand is first transcribed into a DNA strand with the aid of reverse transcriptase.
With the aid of NASBA large amounts of single stranded RNA are generated from either single stranded RNA or DNA or double stranded DNA. When RNA is to be amplified the ssRNA serves as a template for the synthesis of a first DNA strand by elongation of a first primer containing a ssRNA polymerase recognition site. The formed DNA strand in turn serves as the template for the synthesis of a second, complementary, DNA strand by elongation of a second primer, resulting in a double stranded active RNA-polymerase promoter site, and the second DNA serves as a template for synthesis of large amounts of the first template, the ssRNA, with the aid of RNA polymerase.
Detection of the amplified nucleotide sequence is established by hybridizing a complementary detection probe to the amplified nucleic acid. This probe can be labelled and/or immobilized on a solid phase.
Detection of the label can be performed through methods known in the art. Detection of nucleic acids bound through the probe to the solid phase can be done by compounds capable of selective detection of nucleic acids.
As said before the nucleotide sequences can be used for the production of gp100 or one of its epitopes with recombinant DNA techniques. For this the nucleotide sequence must be comprised in a cloning vehicle which can be used to transform or transfect a suitable host cell.
A wide variety of host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence. For example, useful cloning vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids, and wider host range plasmids such as pBR 322, the various pUC, POEM and pBluescript plasmids, bacteriophages, e.g. lambda-gt-Wes, Charon 28 and the M13 derived phages and vectors derived from combinations of plasmids and phage or virus DNA, such as SV40, adenovirus or polyoma virus DNA (see also Rodriquez, R. L. and Denhardt (1988); Lenstra, 1990).
Useful hosts may include bacterial hosts, yeasts and other fungi, plant or animal hosts, such as Chinese Hamster Overy (CHO) cells or monkey cells and other hosts.
Vehicles for use in expression of the peptides will further comprise control sequences operably linked to the nucleic acid sequence coding for the peptide. Such control sequences generally comprise a promoter sequence and sequences which regulate and/or enhance expression levels. Furthermore an origin of replication and/or a dominant selection marker are often present in such vehicles. Of course control and other sequences can vary depending on the host cell selected.
Techniques for transforming or transfecting host cells are quite known in the art (see, for instance, Maniatis et al., 1982 and 1989).
It is extremely practical if, next to the information for the peptide, also the host cell is co-transformed or co-transfected with a vector which carries the information for an MHC molecule to which said peptide is known to bind. Preferably the MHC molecule is HLA-A2.1, HLA-A1 or HLA-A3.1, or any other HLA allele which is known to be present in melanoma patients. HLA-A2.1 is especially preferred because it has been established (Anichini A., 1993) that melanoma cells carry antigens recognized by HLA-A2.1 restricted cytotoxic T cell clones from melanoma patients.
Host cells especially suited for the expression of gp100 are the murine EL4 and P8.15 cells. For expression of gp100 human BLM cells (described by Katano, M., 1984) are especially suited because they already are able to express the MHC molecule HLA-A2.1.
Gp100 or any of its peptides or their nucleotide sequences mentioned above can be used in a vaccine for the treatment of melanoma.
In addition to an immunogenically effective amount of the active peptide the vaccine may contain a pharmaceutically acceptable carrier or diluent.
The immunogenicity of the peptides of the invention, especially the oligopeptides, can be enhanced by cross-linking or by coupling to an immunogenic carrier molecule (i.e. a macromolecule having the property of independently eliciting an immunological response in a patient, to which the peptides of the invention can be covalently linked).
Covalent coupling to the carrier molecule can be carried out using methods well known in the art, the exact choice of which will be dictated by the nature of the carrier molecule used. When the immunogenic carrier molecule is a protein, the peptides of the invention can be coupled, e.g. using water soluble carbodiimides such as dicyclohexylcarbodiimide, or glutaraldehyde.
Coupling agents such as these can also be used to cross-link the peptides to themselves without the use of a separate carrier molecule. Such cross-linking into polypeptides or peptide aggregates can also increase immunogenicity.
Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. phosphate buffer).
Optionally, one or more compounds having adjuvant activity may be added to the vaccine. Suitable adjuvants are for example aluminium hydroxide, phosphate or oxide, oil-emulsions (e.g. of Bayol F(R) or Marcol 52(R)), saponins or vitamin-E solubilisate.
The vaccine according to the present invention can be given inter alia intravenously, intraperitoneally, intranasally, intradermally, subcutaneously or intramuscularly.
The useful effective amount to be administered will vary depending on the age and weight of the patient and mode of administration of the vaccine.
The vaccine can be employed to specifically obtain a T cell response, but it is also possible that a B cell response is elicited after vaccination. If so, the B cell response leads to the formation of antibodies against the peptide of the vaccine, which antibodies will be directed to the source of the antigen production, i.e. the tumor cells. This is an advantageous feature, because in this way the tumor cells are combatted by responses of both immunological systems.
Both immunological systems will even be more effectively triggered when the vaccine comprises the peptides as presented in an MHC molecule by an antigen presenting cell (APC). Antigen presentation can be achieved by using monocytes, macrophages, interdigitating cells, Langerhans cells and especially dendritic cells, loaded with one of the peptides of the invention. Loading of the APC""s can be accomplished by bringing the peptides of the invention into or in the neighbourhood of the APC, but it is more preferable to let the APC process the complete gp100 antigen. In this way a presentation is achieved which mimicks the in vivo situation the most realistic. Furthermore the MHC used by the cell is of the type which is suited to present the epitope.
An overall advantage of using APC""s for the presentation of the epitopes is the choice of APC cell that is used in this respect. It is known from different types of APC""s that there are stimulating APC""s and inhibiting APC""s.
Preferred are the listed cell types, which are so-called xe2x80x98professionalxe2x80x99 antigen presenting cells, characterized in that they have co-stimulating molecules, which have an important function in the process of antigen presentation. Such co-stimulating molecules are, for example, B7, CTLA-4, CD70 or heat stable antigen (Schwartz, 1992).
Fibroblasts, which have also been shown to be able to act as an antigen presenting cell, lacks these co-stimulating molecules.
It is also possible to use cells already transfected with a cloning vehicle harbouring the information for gp100 and which are cotransfected with a cloning vehicle which comprises the nucleotide sequence for an MHC class I molecule, for instance the sequence coding for HLA A2.1, HLA A1 or HLA A3.1. These cells will act as an antigen presenting cell and will present gp100-fragments in the MHC class I molecules which are expressed on their surface. It is envisaged that this presentation will be enhanced, when the cell is also capable of expressing one of the above-mentioned co-stimulating molecules, or a molecule with a similar function. This expression can be the result of transformation or transfection of the cell with a third cloning vehicle having the sequence information coding for such a co-stimulating molecule, but it can also be that the cell already was capable of production of co-stimulating molecules.
In stead of a vaccine with these cells, which next to the desired expression products, also harbour many elements which are also expressed and which can negatively affect the desired immunogenic reaction of the cell, it is also possible that a vaccine is composed with liposomes which expose MHC molecules loaded with peptides, and which, for instance, are filled with lymphokines. Such liposomes will trigger a immunologic T cell reaction.
By presenting the peptide in the same way as it is also presented in vivo an enhanced T cell response will be evoked. Furthermore, by the natural adjuvant working of the, relatively large, antigen presenting cells also a B cell response is triggered. This B cell response will a.o. lead to the formation of antibodies directed to the peptide-MHC complex. This complex is especially found in tumor cells, where it has been shown that in the patient epitopes of gp100 are presented naturally, which are thus able to elicit a T cell response. It is this naturally occurring phenomenon which is enlarged by the vaccination of APC""s already presenting the peptides of the invention. By enlarging not only an enlarged T cell response will be evoked, but also a B cell response which leads to antibodies directed to the MHC-peptide complex will be initiated.
The vaccines according to the invention can be enriched by numerous compounds which have an enhancing effect on the initiation and the maintenance of both the T cell and the B cell response after vaccination.
In this way addition of cytokines to the vaccine will enhance the T cell response. Suitable cytokines are for instance interleukines, such as IL-2, IL-4, IL-7, or IL-12, GM-CSF, RANTES, tumor necrosis factor and interferons, such as IFN-.
In a similar way antibodies against T cell surface antigens, such as CD2, CD3, CD27 and CD28 will enhance the immunogenic reaction.
Also the addition of helper epitopes to stimulate CD4+ helper cells or CD8+ killer cells augments the immunogenic reaction. Alternatively also helper epitopes from other antigens can be used, for instance from heat shock derived proteins or cholera toxin.
Another part of the invention is formed by usage of gp100 reactive tumor infiltrating lymphocytes (TIL""s). In this method the first step is taking a sample from a patient. This is usually done by resection of a tumor deposit under local anaesthesia. The TIL""s present in this specimen are then expanded in culture for four to eight weeks, according to known methods (Topalian, S. L. et al., 1987). During this culture the TIL""s are then checked for reactivity with gp100 or one of the epitopes derived from gp100. The TIL""s which recognize the antigen are isolated and cultured further.
The tumor infiltrating lymphocytes, reactive with gp100, which are obtained through this method, form also of the invention. One such TIL cell line, designated TIL 1200, has been found which specifically reacts with gp100 and its epitopes. This TIL 1200 cell line also expresses the MHC molecule HLA-A2.1. Furthermore expression of TCR xcex1/xcex2, CD3 and CD8 by this cell line has been demonstrated. Furthermore TIL 1200 recognizes transfectants expressing both HLA-A2.1 and gp100.
This TIL 1200 and other TIL""s recognizing gp100 are suited for treatment of melanoma patients. For such treatment TIL""s are cultured as stated above, and they are given back to the patients by an intravenous infusion. The success of treatment can be enhanced by pre-treatment of the tumor bearing host with either total body radiation or treatment with cyclophosphamide and by the simultaneous administration of interleukin-2 (Rosenberg, S. A. et al., 1986).
The TIL""s infused back to the patient are preferably autologous TIL""s (i.e. derived from the patient""s own tumor) but also infusion with allogenic TIL""s can be imagined.
A further use of the TIL""s obtained by the method as described above is for in vivo diagnosis. Labelling of the TIL""s, for instance with 111In (Fisher, 1989) or any other suitable diagnostic marker, renders them suited for identification of tumor deposits in melanoma patients.
Another part of the invention is formed by the T cell receptor (TCR) expressed by gp100 reactive CTLs. As is well known in the art, the TCR determines the specificity of a CTL. Therefore, the cDNA encoding the TCR, especially its variable region, can be isolated and introduced into T cells, hereby transferring anti-tumor activity to any T cell. Especially introduction of such a TCR into autologous T cells and subsequent expansion of these T cells, will result in large numbers of CTL suitable for adoptive transfer into the autologous patient.
Also cells harbouring this T cell receptor can be used for vaccination purposes.
A vaccine can also be composed from melanoma cells capable of expression of gp100. It is possible to isolate these cells from a patient, using anti-gp100 antibodies, such as NKI-beteb, but is also possible to produce such melanoma cells from cultured melanoma cell lines, which either are natural gp100-producers or have been manipulated genetically to produce gp100. These cells can be irradiated to be non-tumorogenic and infused (back) into the patient. To enhance the immunologic effect of these melanoma cells it is preferred to alter them genetically to produce a lymphokine, preferably interleukine-2 (IL-2) or granulocyte-macrophage colony stimulation factor (GM-CSF). Gp100+ melanoma cells can be transfected with a cloning vehicle having the sequence coding for the producti on of IL-2 or GM-CSF.
Infusion of such a vaccine into a patient will stimulate the formation of CTL""s.
Another type of vaccination having a similar effect is the vaccination with pure DNA, for instance the DNA of a vector or a vector virus having the DNA sequence encoding the gp100 antigen or peptides derived therefrom. Once injected the virus will infect or the DNA will be transformed to cells which express the antigen or the peptide(s).
Antibodies to any gp100 peptide, including antibodies to (V)-(W)-(K)-T-W-G-Q-Y-W-Q-V-(L) SEQ ID NO:10, and L-L-D-G-T-A-T-L-R-L SEQ ID NO:4, are also part of the invention.
Monospecific antibodies to these peptides can be obtained by affinity purification from polyspecific antisera by a modification of the method of Hall, R. et al. (1984). Polyspecific antisera can be obtained by immunizing rabbits according to standard immunisation schemes.
Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen. Homogeneous binding as used herein refers to the ability of the antibody species to bind to ligand binding domain of the invention.
The antibody is preferably a monoclonal antibody, more preferably a humanised monoclonal antibody.
Monoclonal antibodies can be prepared by immunizing inbred mice, preferably Balb/c with the appropriate protein by techniques known in the art (Kxc3x6hler, G. and Milstein C., 1975). Hybridoma cells are subsequently selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium such as Dulbecco""s modified Eagle""s medium (DMEM). Antibody producing hybridomas are cloned, preferably using the soft agar technique of MacPherson (1973). Discrete colonies are transferred into individual wells of culture plates for cultivation in an appropriate culture medium. Antibody producing cells are identified by screening with the appropriate immunogen. Immunogen positive hybridoma cells are maintained by techniques known in the art. Specific anti-monoclonal antibodies are produced by cultivating the hybridomas in vitro or preparing ascites fluid in mice following hybridoma injection by procedures known in the art.
It is preferred to use humanized antibodies. Methods for humanizing antibodies, such as CDR-grafting, are known (Jones, P. T. et al., 1986). Another possibility to avoid antigenic response to antibodies reactive with polypeptides according to the invention is the use of human antibodies or fragments or derivatives thereof.
Human antibodies can be produced by in vitro stimulation of isolated B-lymphocytes, or they can be isolated from (immortalized) B-lymphocytes which have been harvested from a human being immunized with at least one ligand binding domain according to the invention.
Antibodies as described above can be used for the passive vaccination of melanoma patients. A preferred type of antibodies for this kind of vaccine are antibodies directed against the above-mentioned peptides presented in connection with the MHC molecule. To produce these kind of antibodies immunization of peptides presented by APC""s is required. Such an immunization can be performed as described above. Alternatively, human antibodies to peptide-MHC complexes can be isolated from patients treated with a vaccine consisting of APC""s loaded with one of said peptides.
The antibodies, which are formed after treatment with one of the vaccines of the invention can also be used for the monitoring of said vaccination. For such a method serum of the patients is obtained and the antibodies directed to the peptide with which has been vaccinated are detected. Knowing the antibody titre from this detection it can be judged if there is need for a boost vaccination.
Specific detection of said antibodies in the serum can be achieved by labelled peptides. The label can be any diagnostic marker known in the field of in vitro diagnosis, but most preferred (and widely used) are enzymes, dyes, metals and radionuclides, such as 67Ga, 99mTc, 111In, 113mIn, 123I, 125I or 131I.
The radiodiagnostic markers can be coupled directly to the peptides of the invention or through chelating moieties which have been coupled to the peptide directly or through linker or spacer molecules. The technique of coupling of radionuclides to peptides or peptide-like structures is already known in the field of (tumor) diagnostics from the numerous applications of labelled antibodies used both in in vivo and in in vitro tests.
Direct labelling of peptides can for instance be performed as described in the one-vial method (Haisma, 1986). A general method for labelling of peptides through chelators, with or without linker or spacer molecules, has for instance been described in U.S. Pat. Nos. 4,472,509 and 4,485,086. Chelators using a bicyclic anhydride of DTPA have been disclosed in Hnatowich, D. J. et al. (1983). Coupling through diamide dimercaptide compounds has been disclosed in EP 188,256.